In the past year, we have continued to characterize the single known paternal-effect-lethal (PEL) protein in C. elegans, SPE-11, through structure function analysis. SPE-11 is a 299 amino acid protein with no obvious protein domains. SPE-11 does have five predicted nuclear localization sequences (NLS) and localizes to germ-line nuclei of the male. To determine which of the five predicted NLSs are important for SPE-11 localization and if proper localization is important for SPE-11 function, each NLS will be systematically mutated from the GFP::SPE-11 reporter construct that was generated in our lab. The mutant constructs will be used to create transgenic animals, which will then be analyzed for proper SPE-11 localization and rescue of a spe-11 deletion mutant. NLS mutants that show mislocalization will be evaluated to determine their effects on early embryonic events. Thus far, we have created two single NLS mutant constructs and one double NLS mutant construct. A single integrated line harboring the GFP::SPE-11 NLS1 mutant has been generated. This mutant line is able to rescue the embryonic lethality of the spe-11 deletion mutant and localizes correctly in the male germ line. We have also generated a single integrated line with the GFP::SPE-11 NLS5 mutant. This line localizes correctly in the male germ line but fails to rescue the spe-11 deletion mutant. Because the NLS5 mutant localizes correctly, it does not reveal whether localization is important for function. However, it is unable to rescue the spe-11 deletion mutant, suggesting that these amino acid residues are important for the function of SPE-11. We are continuing our efforts to make integrated transgenic lines of other NLS mutants. Interestingly, NLS4 and NLS5 reside in the C-terminus of the SPE-11 protein. Curiously, the majority of existing spe-11 mutant alleles result in C-terminal truncations of the SPE-11 protein, suggesting that the C-terminus is very important for function. To determine which part of the C-terminus is essential for early embryogenesis, we are creating constructs with truncated versions of SPE-11 and generating transgenic lines as mentioned previously. The mutant allele with the longest predicted SPE-11 protein has an early stop codon at amino acid 244. We have created two constructs with truncated versions of SPE-11 (GFP::SPE-11 1-289 and GFP::SPE-11 1-294) and generated transgenic lines. Analysis of these two mutant lines has found that both fail to rescue the embryonic lethality of the spe-11 deletion mutant suggesting that even the most C-terminal end of the protein (i.e. the last five amino acids) are essential for function. We are continuing to test additional SPE-11 NLS mutants and whether SPE-11 variants with specific regions deleted are capable of rescue. We have also pursued a study to identify additional PEL mutants. Previous genetic screens identified embryonic lethal mutants that could be rescued by mating with wild-type males, thus indicating a sperm defect. The ultimate test is to show that mutant males sire dead progeny when mated to wild type hermaphrodites (or females). We have found one such mutant, mel-15. Initial analysis confirms that sperm from mel-15 mutant males produce dead embryos, even when fertilizing wild-type oocytes. In addition, characterization of early embryonic events indicates fertilization is successful and that sperm components (e.g. centrosomes) are transmitted to the embryo, but that mel-15 male sperm lack DNA. Preliminary characterization of meiotic events in mel-15 male germ lines points to a defect in chromosome segregation during meiosis I. We are currently in the process of determining the molecular identity of mel-15. Once confirmed, we will further characterize the mel-15 phenotypes, determine expression patterns, and start suppression screens to identify other genes in the pathway. We hope to also determine whether mel-15 functions with spe-11 or is involved in a distinct process.